Immunity to human cytomegalovirus (HCMV) is complex, and requires both humoral immune responses (predominately antibody to envelope glycoproteins) and cellular immunity (CD4+ and CD8+ responses to multiple structural/regulatory proteins). Because of lifelong disabilities caused by congenital HCMV infection, understanding the host defense determinants that protect the developing fetus is critical, toward the goal of developing an effective preconception vaccine. Clinical trials of an adjuvanted glycoprotein B (gB) vaccine showed some promise in young women of childbearing age, but waning immunity and modest efficacy (~50%) necessitate consideration of other strategies. A key to protection against HCMV disease is the development of MHC class I restricted, cytotoxic CD8+ T-lymphocyte responses. The ability to quantify both the effector functions and cytokine profiles of these cells is a critical aspect of the evaluation of the effectiveness of HCMV vaccines. In particular, measuring the elaboration of interferon gamma (IF-?) by T cells following vaccination is vital, since this cytokine plays a key role in protectio. The guinea pig cytomegalovirus (GPCMV) model of congenital infection provides a useful system for evaluating vaccine-mediated protection, but, unfortunately, evaluation of T cell responses has been problematic, largely due to a lack of immunological assays and reagents for guinea pig research. To address this deficiency, aim 1 of this application proposes to develop a novel IF-? ELISPOT assays for the guinea pig, using a panel of recently developed monoclonal antibodies. In aim 2, this assay will be used to examine and validate, using overlapping peptide libraries, the response to a known GPCMV T-cell target, GP83 (HCMV pp65 homolog). The precise peptide epitope(s) critical in the GP83- specific response of GPCMV-infected inbred strain 2 guinea pigs will be mapped. In addition, we will use ELISPOT to interrogate, with peptide libraries, the T cell response to GPCMV ORFs GP32, GP48, GP48a, GP55 (gB homolog), GP82, GP99, GP122 (IE2), and GP123 (IE1). These ORFs are hypothesized to be important in the guinea pig cellular response to GPCMV infection, since: 1) T-cell responses are frequent following HCMV infection, as well as other CMVs; 2) these ORFs elicit both CD4+ and CD8+ responses in the setting of HCMV infection; and 3) the ORFs are well-conserved in the GPCMV genome. These experiments, utilizing the R03 mechanism, will support development of new research technologies/methodologies, allow pilot and feasibility studies of the guinea pig T cell response, and enable identification of specific peptide epitopes important in GPCMV infection. These studies will provide novel, new information about the cellular immune response to GPCMV; will facilitate development of an important assay heretofore unavailable for guinea pig research; and will have implications for design of improved HCMV vaccines. Eventually, polyvalent T-cell vaccines aimed at augmenting immunity conferred by antibody-based glycoprotein vaccines may improve prospects for protecting infants against congenital HCMV infection, a major and unmet public health priority.